1. Field of the Invention
The present invention relates to a method of isolating and proliferating autologous, antigen-specific CD8+ T cells using anti-4-1BB antibodies.
2. Description of the Related Art
Research into the treatment of cancer using a patient's autoimmune system has been going on for a long time. The main targets of such treatment are dendritic cells (DCs) and CD8+ T cells (O'Neill D W et al., Manipulating dendritic cell biology for the active immunotherapy of cancer, Blood, 104, pp. 2235-2246, 2004). The DCs are potent antigen-presenting cells that are able to induce strong immunity to a labeled antigen, and thus have been studied as promising immuno-therapeutics candidates (Preynat-Seauve O et al., Tumor-infiltrating dendritic cells are potent antigen-presenting cells able to activate T cells and mediate tumor rejection, J Immunol., 176, pp. 61-67, 2006). However, recent reports indicate that functional diversity of the DC brings about suppression of immunity as well as enhancement of immunity that has been expected to occur in a living body (Zhang X et al., CD4-8-dendritic cells prime CD4+ T regulatory 1 cells to suppress antitumor immunity, J immunol, 175, pp. 2931-2937, 2005). It is expected that cancer treatment using DCs is also directed to strong activation of CD8+ T cells, and CD8+ T cell-induced antitumor immunity may suppress a recurrence of the tumor cell by producing a memory T cell.
The CD8+ T cells have less probability to exhibit unexpected side-effects since they have a relatively simpler function than other cells, for example, DCs, CD4+ T cells and NK cells. The present inventor has been conducting a study of 4-1BB (CD137), an immunoregulatory protein, for a long period of time, and has come a long way in analyzing its characteristics and immunoregulation. The in vitro and in vivo characteristics of a 4-1BB stimulus are as follows. From results of the long in-vitro experiment on CD4+ and CD8+ T cells, it can be noted that CD8+ T cells stimulated by 4-1BB exhibit strong cytotoxicity, high expression of IFN-γ, and inhibition of activation-induced cell death (AICD) (Hurtado J C et al., Signals through 4-1BB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death, J Immunol., 158, pp. 2600-2609, 1997). Based on such characteristics of the 4-1 BB stimulus, which are appropriate for cancer treatment, a therapeutic effect on cancer using anti-4-1BB mAb was proven through an animal model (Wilcox R A et al., Provision of antigen and CD137 signaling breaks immunological ignorance, promoting repression of poorly immunogenic tumors, J Clin Invest., 109, pp. 651-659, 2002).
In response to effects of a cellular 4-1BB chain reaction, a chain reaction of the CD4+ T cell exhibits inductive expression by TCR activation, enhanced proliferation, cell cycle progression, AICD inhibition, production of Th1 cytokines such as IL-2, IFN-γ and IL-4, and increased memory formation. Further, a chain reaction of the CD8+ T cell exhibits inductive expression by TCR activation, enhanced proliferation, AICD inhibition, production of Th1 cytokines such as IL-2, IFN-γ and TNF-α, enhanced cytotoxic function, and increased memory formation.
In response to effects of an in vivo 4-1BB chain reaction, a cancer model exhibits an increased lifespan of a tumor-bearing mouse and cancer growth inhibition by enhanced CD8+ T reaction, an autoimmune disease model exhibits inhibition of disease progression by suppression of an antigen-specific CD4+ T cell, a viral infection model exhibits virus propagation inhibition by enhanced CD4+ T cell reaction, Listeria infection model exhibits protection of a Listeria-infected mouse, a GVHD model exhibits GVHD prevention by inhibition of CD4+ T cell reaction, and a transplantation model exhibits promotion of transplant rejection.
4-1BB, an inductive costimulatory molecule, is expressed in an activated CD8+ T cell. Thus, when CD8+ T cell is stimulated by a peptide, 4-1BB is generally expressed in the CD8+ T cell specifically activated by the peptide, and thus the cells may be isolated. By such a principle, only an activated and peptide-specific CD8+ T cell is isolated from peripheral blood mononuclear cells (PBMCs) which have been isolated from a patient's blood. The isolated CD8+ T cell is to be injected back into the patient after being proliferated as much as necessary using IL-2. An MHC-class I pentamer is an agent distinguishing an antigen-specific CD8+ T cell by forming a complex with a peptide to specifically bind to TCR.
Most people have been infected by CMV during their growth, and thus have immunological memories against CMV in a T cell, which has strong antigenicity, thereby inducing proliferation of antigen-specific CD8+ T cells. Accordingly, MHC class I pentamers for detecting such CMV-specific CD8+ T cells have been also developed, and are now commercially available.
Most people have been infected by Epstein-Barr virus (EBV), the most common virus, in childhood, but generally exhibit no symptoms. However, they still have immunological memories. The EBV also relates to various types of human malignant tumors, which is a main cause of African Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC). In addition, EBV is reportedly linked to many diseases related to T cell dysfunction due to AIDS and immune suppressive drugs after transplantation, hairy leukoplakias, B cell lymphoproliferative diseases (EBV-LPDs), Hodgkin's lymphomas, gastric carcinomas, some breast carcinomas, etc. Since many people have memory T cells against the EBV, which is a viral antigen, the EBV can induce a stronger immune response than common antigens. Due to its high reactivity of CD8+ T cells and many patients, the EBV serves as a good test model for development of CTL cell therapy products using peptides and anti-4-1 BB antibodies.
After attempts to establish a therapeutic method of producing CD8+ T cell therapy products which are essential for eliminating tumor cells and specifically response to cytomegarovirus (CMV) pp65, the present invention was completed by providing methods of massively isolating and proliferating CD8+ T cells specifically responding to CMV pp65 and confirming that the CD8+ T cells yielded by these methods can be used to treat cancer without any side effects.